The binding of streptonigrin (SN) to nucleic acids was studied in the presence of reducing agents and metals. Incubation of chemically reduced SN with DNA in vitro resulted in irreversible binding and complexes containing 1 mole of SN per 250 nucleotides were obtained. The presence of Zn2 ions increased this binding considerably to give complexes containing 1 mole of SN per 80 nucleotides. On the other hand, Mg2 ions decreased this binding. More drug was bound to denatured DNA than to native DNA. Maximum binding was obtained when SN was reduced in the presence of DNA. Increased binding was also obtained when the fully reduced SN was incubated with DNA. Furthermore, enzymatically activated SN binds to DNA to a greater degree than SN activated by chemical systems. Studies with synthetic polynucleotides in the presence of Zn2 ions suggested that while SN has a high affinity for guanine, cytosine and adenine also serve as excellent substrates. These studies indicate that the active intermediate that binds to nucleic acids is unstable and may be derived from the fully reduced drug. These in vitro studies further suggest that Zn2 ions play an important role in the binding of SN to DNA and may have implications for the biological actions of SN if similar reactions occurred in vivo.